Abiotic Nitrite Incorporation into Organic Matter in Volcanic and Non-Volcanic Soil Within Rainforest Ecosystems

Abstract

Understanding nitrogen (N) retention mechanisms in pristine humid temperate rainforest soils is critical for effective ecosystem management and nutrient conservation. The potential abiotic transformation of nitrite (NO₂⁻) into organic N forms in the absence of microbial activity in these ecosystems remains largely unexplored, despite its role in mitigating N leaching. This study focuses on the abiotic incorporation of nitrite (NO₂⁻) into dissolved organic nitrogen (DON) under anoxic conditions, a mechanistic step not directly evaluated in previous research, which employed ¹⁵N-labelled nitrate (NO₃⁻). To address this gap, we used ¹⁵N-labelled NO₂⁻ at 5 and 15 mg L⁻¹ in a lab incubation study under anoxic conditions to trace the contribution of abiotic nitrite transformation to organic N formation in organic matter-rich soils from temperate rainforests developed on both volcanic and non-volcanic parent materials. The added ¹⁵N declined rapidly after 15 min by 52% and 60% in both soil solutions, while it started to form labelled DON, increasing by 11% and 34%, after five days of incubation, with the highest accumulation at 15 mg L⁻¹ of ¹⁵N-NO₂⁻. These results show that up to 77% of the added ¹⁵N-NO₂⁻ can be abiotically incorporated into the DON of unpolluted old-growth temperate rainforest, whether developed on volcanic or non-volcanic soils. Nitrogen input has a stronger effect than soil parent material from which the soils originate. This reveals the natural resilience of unpolluted temperate rainforests to N loss, with implications for long-term ecosystem stability and nutrient cycling.

1. Introduction

A comprehensive understanding of nitrogen (N) retention in humid temperate rainforest soils is essential for effective ecosystem management and nutrient conservation. However, biotic and abiotic factors that shape the stabilization of N in temperate forests are still not fully understood [1,2]. Recent studies indicate that dissolved organic nitrogen (DON) formation and persistence may occur independently of biological activity, particularly in soils of volcanic origin classified according to Soil Survey Staff 2014 [3] as Andisols or as Andosols according to the WRB 2022 [4], which are characterized by an extremely high soil organic matter (SOM) content due to the reactive short-range order minerals such as allophane, imogolite and ferrihydrite [2]. These soils exhibit conservative N cycling even under exceptionally humid conditions and are notably rich in iron [5], a key factor in redox-mediated transformations involving nitrate and nitrite (NO₂⁻) [6,7].

Long-term studies in pristine old-growth rainforests reveal that N is primarily lost in organic (~95%) forms, rather than in mineral forms [8]. Tracer ¹⁵N showed that ~65% of the labelled N was retained in stable SOM after two years of field experiments [9]. Later, in several undisturbed temperate forests in the Southern Hemisphere (Chile and Argentina), N losses were dominated by DON rather than mineral N, which is the opposite of the pattern observed in the Northern Hemisphere [10]. Most of these losses occurred in Andisols [3], although soils derived from metamorphic rock classified as Ultisol [3] or equivalent to Acrisol [4] were also included where anaerobic conditions favored dissimilatory nitrate reduction to ammonium (DNRA) [2]. Matus et al. [6] demonstrated that 24% and 37% of the added ¹⁵N-NO₃⁻ was incorporated into sterilized DON from an Andisol incubate under anoxic conditions within just 15 min. This finding suggests that nitrosation could be a likely mechanism. However, they did not investigate the potential role of NO₂⁻ as an intermediate in this process. To ensure accurate measurements of mineral N, they directly sampled the soil solution using an automated sampling device coupled to a quadrupole mass spectrometer, thereby avoiding the redox interference that occurs when using a traditional copper–cadmium column for mineral N determination.

The nitrosation reaction pathway is as follows:

$${\mathrm{N}\mathrm{O}}_2^{-}+{\mathrm{H}}^{+}\rightleftharpoons {\mathrm{H}\mathrm{N}\mathrm{O}}_2$$

(1)

$${\mathrm{H}\mathrm{N}\mathrm{O}}_2+\mathrm{H}\rightleftharpoons {\mathrm{N}\mathrm{O}}^{+}+{\mathrm{H}}_2\mathrm{O}$$

(2)

$$\mathrm{A}\mathrm{r}\mathrm{O}\mathrm{H}+{\mathrm{N}\mathrm{O}}^{+}\to \mathrm{O}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{c}-\mathrm{N}.$$

(3)

Nitrosyl ion NO⁺ reacts through electrophilic substitution in the aromatic ring (ArOH) to form nitrosophenols [11]. The first evidence for NO₂⁻ incorporation into SOM was obtained early by Fuhr and Bremner [12,13]. By applying Na¹⁵NO₂, they observed that a portion of nitrite-N was retained by organic matter with circumneutral pH under aerobic conditions. This effect increased exponentially with rising soil organic carbon (SOC) content and declined as soil pH increased [12]. However, the transformation of NO₂⁻ into DON in the soil solution of soils with very high SOC content remains unexplored. The role of abiotic N retention in the formation or stabilization of DON is still uncertain [14,15,16,17,18,19], particularly in humid temperate rainforest ecosystems. This includes volcanic soil (Andisols [3]) derived from recent tephra in the piedmont of the Andes range, as well as non-volcanic soil (Ultisols [3])) from the summit plateau, which is derived from metamorphic rocks in the Coastal range. In these ecosystems, losses of DON can exceed those of NO₃⁻ leaching [10] and it was recently demonstrated that land use has a minimal effect compared with the key role of parent materials in nitrite dynamics [20]. We found only one study, by Boudot et al. [14], that reported on the incorporation of NO₂⁻ into SOM in an andic soil.

The transformation of NO₂⁻ into DON is particularly important in supporting the Ferrous Wheel Hypothesis (FWH), which may explain the low mineral N leaching and the comparatively high DON losses observed in the watersheds of southern Chile. The FWH proposes that in anaerobic microsites in the soil, Fe(II) reduces nitrate to nitrite, after which Fe(II) is re-oxidized to Fe(III), probably in an aerobic phase. This redox cycling facilitates the formation of DON via nitrosation reactions [21,22].

This study expands on the previous one [6], which did not directly examine the transformation of NO₂⁻ into DON. The objective was to investigate the incorporation of N-NO₂⁻ into DON, in the soil solution extracted from the Ah horizon of an Andisol [3]. Additionally, it explores non-volcanic soil, an Ultisol [3], both soils having been developed under a humid temperate rainforest. This study tested the hypothesis that NO₂⁻ contributes to the formation of organic N in organic matter-rich soils of contrasting origins from temperate rainforests.

2. Materials and Methods

2.1. Soil Sampling

Two soil types were sampled at similar latitudes (40°38′ S and 72°5′ W, 800–1000 m a.s.l.). Andisols as classified as by the Soil Survey Staff [3] or as Andosols by the WRB [4] were collected from Puyehue National Park (Andes Mountains), and Ultisol or Acrisol soils (40°11′ S and 73°28′ W, 1000 m a.s.l.) were collected from Alerce Costero National Park (Coastal range). Andisols are characterized by native vegetation such as Nothofagus betuloides (Mirb.) Oerst., and Ultisols are characterized by different species: Weinmannia trichosperma, Cav., Nothofagus nitida (Phil.) Krasser, Saxegothaea conspicua Lindl., Laureliopsis Philippiana (Looser) Schodde., Podocarpus nubigena Lindl., and Drimys winteri J.R.Forst and G.Forst.

The selected forest soils are ideal representatives to study the role of SOM in N dynamics. Our choice was supported by the idea that regions with naturally low inputs of N from atmospheric deposition and N fixation, which were floristically stable throughout the Holocene/late Pleistocene [10], represent long-term N conservation through abiotic and biotic soil processes. These ecosystems have common characteristics, such as high organic C content due to low temperatures < 10 °C, high precipitation (>4000 mm), and significant losses of DON in streams [9,10]. The parent material of Andisols [3] is dominated by basaltic scoria ejecta and short-range order minerals, including allophane, imogolite, and ferrihydrite from the piedmont of the Andes Mountains, characterized by a disproportionate amount of SOC, 110.00 ± 8.00 g kg⁻¹, and total N content of 6.00 ± 0.30 g kg⁻¹, with a moderately low pH 5.7 ± 0.13. The non-volcanic Ultisols are marked by a strong presence of illite and kaolinite [23], which developed in situ at the summit plateau of ancient (Palaeozoic) metamorphic (mica-schists) tectonic uplifts from Coastal range [24]. These soils are typical of metamorphic complexes [25] with an SOC of 99.00 ± 3.00 g kg⁻¹, total N of 4.0 ± 0.0 g kg⁻¹ and pH of 4.5 ± 0.2.

Sampling involved collecting a composite mineral soil sample from the same soil series with the organic horizon removed, using a stainless-steel cylinder (4.6 diameter and 8 cm in length). Each composite sample was composed of 5 to 8 subsamples taken from locations 30 m apart at each site. All soil samples were transported under cool conditions. Once in the laboratory, the samples were cleaned, air-dried, and then sieved to a size of 2 mm for further analysis.

2.2. Dissolved Organic Nitrogen

Approximately 1.5 kg of each soil was divided into four portions (four replicates). Dissolved organic N was extracted from approximately 120 g of field-moist soil, equivalent to 100 g dry weight, which was suspended in 200 mL of Milli-Q water. The mixture was agitated at 180 rpm for 10 min at room temperature, followed by centrifugation at 4000 rpm. The resulting supernatant was passed through a 0.40 mm polycarbonate membrane filter. In total, we collected 2.5 L from each soil. A detailed characterization of DON is provided in

Table 1. Mean and standard error of the mean properties of dissolved organic nitrogen (DON) extracted from 5–15 cm volcanic and non-volcanic mineral soil samples from temperate rainforests.

Analysis	Units	Volcanic	Non-Volcanic
DON 1	mg L−1	26.20 ± 4.93	5.29 ± 0.01
DOC 2	mg L−1	35.62 ± 0.25	23.78 ± 0.36
EC 3	dS m−1	1.90 ± 0.06	2.00 ± 0.08
pHw 4	unitless	4.30 ± 0.04	3.80 ± 0.05
N-NH4+	mg L−1	2.00 ± 0.77	2.53 ± 1.01
N-NO3−	mg L−1	0.00 ± 0.00	0.04 ± 0.04
N-NO2−	mg L−1	0.00 ± 0.00	0.00 ± 0.00

¹ dissolved organic nitrogen, ² dissolved organic carbon ³ electrical conductivity; ⁴ water pH.

.

2.3. Dissolved Organic Nitrogen Sterilization

About 0.8 L of soil solution extracted from each soil was immediately frozen and transported to Radeberg and sterilized with γ-irradiation (10 h at 53 kGy) at Synergy Health Radeberg GmbH center, Radeberg, Germany.

2.4. Analytical Methods

The pH was measured in a soil–water ratio of 1:2.5 using a WTW inoLab pH 7110 pH meter equipped with a SenTix 21 pH electrode, Xylem Analytics Germany Sales GmbH and Co. KG, Weilheim, Germany. The same instrument was used to measure the electrical conductivity in the same suspension. Total N and C concentrations in the freeze-dried DOM were determined using an elemental analyzer (Multi N/C 2100 S, Analytik Jena, Jena, Germany). The total soil organic C was determined using TOC-VCSH (Shimadzu, Kyoto, Japan), and total N was measured using Kjeldahl distillation (VELP, Usmate, Italy).

2.5. Microcosm Experiment

An 100 mL aliquot of sterilized DOM was transferred into autoclaved 500 mL air-tight incubation flasks equipped with a septum for liquid sampling. All flasks were flushed with argon for 45 min at a continuous flow rate of 5–10 mL min⁻¹ to achieve oxygen-free conditions (Eh < 300 mV at pH < 4.3, electrode PCE-228-R, (PCE Instruments, Meschede, Germany)) [26]. A 1 mL solution containing either 5 or 15 mg L⁻¹ of ¹⁵N-NaNO₂ (99 atom%, Sigma-Aldrich, Steinheim, Germany) was added at a 10% fraction, resulting in final concentrations of 0.50 and 1.50 mg L⁻¹ of ¹⁵N. A control flask containing only Milli-Q water was included in the experiment. In total, 48 sterilized flasks (32 ¹⁵N-labelled and 16 control soil samples) for each soil were incubated, including a control (four replicates). The incubation was performed at 38 °C to accelerate the reaction. Sampling times were set at 15, 60, 1440, and 7200 min (five days) after the addition of the labelled N. After each sampling, the flasks were purged with argon again. At each sampling interval, a 25 mL extract was taken, and 10 mL of this solution was immediately freeze-dried for total N and isotopic ¹⁵N signal determination (see below). The remaining 15 mL was used to measure ammonium (NH4⁺), nitrite (NO₂⁻), and nitrate (NO₃⁻).

2.6. Labelled Nitrogen Concentration

The total N and total nitrogen-15 in DON were measured using elemental analysis–isotope ratio mass spectrometry (EA-IRMS), which was coupled with a Delta Plus system from Finnigan MAT in Bremen, Germany. Mineral N was measured using an automated SPIN-MIMS system, which determines the ¹⁵N concentration of ammonium (NH4⁺), nitrite (NO₂⁻), and nitrate (NO₃⁻) after converting the aqueous solution into N gas (N₂ and NO). All gases were then analyzed using a quadrupole mass spectrometer GAM 200 (InProcess, Bremen, Germany) [27]. The labelled N in DON was calculated as follows:

$${\mathrm{N}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}=\mathrm{D}\mathrm{O}\mathrm{N}+{\mathrm{N}}_{\mathrm{m}\mathrm{i}\mathrm{n}}$$

(4)

$${\mathrm{N}}_{\mathrm{m}\mathrm{i}\mathrm{n}}={\mathrm{N}\mathrm{H}}_4^{+}+{\mathrm{N}\mathrm{O}}_3^{-}+{\mathrm{N}\mathrm{O}}_2^{-}$$

(5)

$$\mathrm{D}\mathrm{O}\mathrm{N}={\mathrm{N}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}-{\mathrm{N}}_{\mathrm{m}\mathrm{i}\mathrm{n}}$$

(6)

where N_total is the labelled total N concentration analyzed in the DON freeze-dried extract with EA-IRMS, and N_min is the labelled mineral ¹⁵N concentrations measured using SPIN-MIMS and a quadrupole mass spectrometer.

2.7. Data Analysis

The normality of the variables of mineral N-NO₃⁻, N-NO₂⁻, N-NH₄⁺, N-total, and their corresponding ¹⁵N values were assessed. If the skewness was 0.50 or higher (indicating a non-Gaussian distribution), the values were log-transformed [28]. The homogeneity of variances was evaluated using sphericity tests. A MANOVA was conducted to analyze the effects of the amount of ¹⁵N added and the incubation time in the microcosm [4]. The significance level was set at p < 0.05.

3. Results

3.1. Soil Solution Characterization

The initial chemical composition of soil solutions prior labelled N addition differed between volcanic and non-volcanic temperate forest soils (Table 1). Total N concentrations were higher in DON of non-volcanic soils (27.17 ± 0.63 mg L⁻¹) than in volcanic soils (22.71 ± 2.42 mg L⁻¹). Similarly, the concentration of DON was slightly greater in non-volcanic soils (24.60 ± 0.06 mg L⁻¹) compared to volcanic soils (20.71 ± 1.64 mg L⁻¹). The DON of volcanic soils exhibited higher pH values (4.30 ± 0.04) than non-volcanic soils (3.80 ± 0.05). Electrical conductivity, used as a proxy for total ionic concentration, was comparable across the DON from both soils, <2.00 ± 0.10 ds m⁻¹. The N-NH₄⁺ concentrations were higher compared to the lower N-NO₃⁻ and non-detectable N-NO₂⁻ concentrations.

3.2. Nitrite Transformation to Organic Nitrogen Forms

The percentage of labelled N recovered in the DON of volcanic soil from 5 and 15 mg L⁻¹ addition fluctuated between 55% and 105% during 5 days of incubation and between 61% and 86% in DON from non-volcanic soil (Figure 1).

After 15 min, the ¹⁵N-nitrite decreased by 49% for 5 mg L⁻¹ in volcanic soils and remained stable (41%–51%) to the end of incubation (p > 0.05) (Figure 2a). As ¹⁵N from NO₂⁻ declined, labelled DON steadily increased up to 0.27 mg L⁻¹, i.e., 55% of the total added (Figure 2b). At 15 mg L⁻¹, a similar pattern was observed, with a more marked decrease of approximately 60% after 15 min of incubation, followed by a relatively stable concentration (Figure 2c). Dissolved organic N gradually increased, although to a lesser extent than at the lower N level, reaching up to 0.43 mg L⁻¹, representing 29% of the addition. In the final sampling, a further decline in ¹⁵N was observed (Figure 2d).

In non-volcanic soils, ¹⁵N-NO₂⁻ exhibited a more pronounced decline of 0.11 mg L⁻¹, equivalent to 77% of added N (Figure 3a). Meanwhile, the labelled DON increased gradually, reaching 57% of the added N, a proportion similar to that observed in volcanic soils (Figure 2b). At the higher N level, ¹⁵N-nitrite decreased by 21% (Figure 3c), while ¹⁵N-DON increased strongly to 35% (p < 0.05), followed by a subsequent decline (Figure 3d).

In general, soils with high N levels tended to show a higher ¹⁵N-DON concentration, while the amount of labelled N-NO₃⁻ and N-NH₄⁺ was barely detectable (Figure 2 and Figure 3). Over the entire sampling period, the average low-level ¹⁵N incorporation into DON was similar for volcanic and non-volcanic soils (p > 0.05), with values of 0.20 ± 0.09 mg L⁻¹ and 0.23 ± 0.03 mg L⁻¹, respectively. At the higher ¹⁵N level (15 mg L⁻¹), the corresponding values were 0.28 ± 0.15 mg L⁻¹ and 0.29 ± 0.15 mg L⁻¹, indicating that the ¹⁵N concentration, rather than the parent material, primarily controls DON formation. Although there were no average differences in DON formation between the two soils, the volcanic soil showed a higher concentration of DON at 60 min during incubation (Figure 3d), consistent with the initial higher dissolved organic carbon and nitrogen levels (Table 1).

4. Discussion

This study demonstrated the conversion of NO₂⁻ into DON under sterilized and anoxic conditions after five days of incubation for both Andisol [3] (Figure 2) and Ultisol [3] (Figure 3) soils. This is important for two reasons: (i) the abiotic pathway may help explain the minimal loss of mineral N, compared to the significant leaching of DON observed in the temperate rainforest watersheds of southern Chile [9,10]; (ii) it represents a crucial step in supporting the Ferrous Wheel Hypothesis (FWH) proposed by Davidson et al. [21,22], independent of the parent material from which soils originate. This hypothesis suggests that Fe(II) reduces NO₃⁻ to NO₂⁻ in anoxic microsites, which leads to the formation of DON while regenerating Fe(III).

Between 21% and 77% of the added N-NO₂⁻ disappeared in the soil solution, where labelled DON increased between 29% and 57% in both soils (Figure 2 and Figure 3). Nitrite immobilization in SOM was first observed in early studies [12,13,29]. However, the conversion of NO₃⁻ through NO₂⁻ to organic forms is still a subject of debate and has only rarely been evaluated [30]. Only one study on andic soil demonstrated that NO₂⁻ was incorporated into the organic fraction through a chemical process involving its self-decomposition and subsequent fixation onto organic matter. Nonetheless, the underlying mechanisms for this process remains unresolved [14].

The results suggest that SOM, not parent material, drives DON formation, as both soils produced similar amounts, Andisol [3] with amorphous clays and Ultisol [3] with more crystalline clays such as kaolinite and illite [23]. Throughout the sampling period, the average ¹⁵N incorporation into DON at both lower and higher concentrations of labelled nitrite added was comparable between volcanic and non-volcanic soils (p > 0.05). Fhür and Bremner [12] reported a ¹⁵N–NO₂⁻ to SOC ratio was ~0.006; whereas, in the current study, the average ¹⁵N-NO₂⁻ (as shown in Figure 2 and Figure 3) divided by dissolved organic carbon (as indicated in Table 1) across all sampling times and labelled doses yielded higher ratios, 0.012 ± 0.01 in volcanic and 0.023 ± 0.03 in non-volcanic soil, likely due to minimal mineral interference at a very low pH, enhancing abiotic nitrite–organic matter reactions. The lithological and chemical characteristics likely contributed little to the organic carbon and nitrogen content. The grey schist and metabasalts near our study sites at Bahía Mansa indicate a sedimentary–volcanic sequence formed in a marginal basin environment [24]. Such low-grade metamorphic complexes typically contain low levels of organic carbon and nitrogen [24,25].

On the other hand, the addition of ¹⁵N-NO₂⁻ did not result in a noticeable production of labelled N-NH₄⁺ and N-NO₃⁻. Consequently, there was an overall recovery of 74%, and the remaining 26% may be attributed to gaseous N losses (N₂O) (Figure 2 and Figure 3). However, gaseous losses previously observed in sterilized DON from volcanic soils [6] were not measured in the current experiment. Although recent studies highlight the parent material as a stronger driver of abiotic N₂O production than land use [20], this was not supported by our findings.

5. Conclusions

This study shows that up to 77% of added ¹⁵N-NO₂⁻ was converted to DON in both volcanic and non-volcanic unpolluted old-growth temperate rainforest soils. We provide strong evidence that the abiotic conversion of NO₂⁻ to DON can occur under sterilized, anoxic conditions in both soil types. These findings support the Ferrous Wheel Hypothesis, highlighting a redox-mediated mechanism by which Fe(II) reduces NO₃⁻ to NO₂⁻ and NO₂⁻ is subsequently incorporated into organic matter as DON. Consistent DON formation from soils across different parent materials suggests that the SOM content plays a greater role than mineral composition in this abiotic pathway. The ¹⁵N–NO₂⁻ to DOC ratios, which were 0.012 ± 0.01 in volcanic and 0.23 ± 0.03 in non-volcanic soils, were much higher than previously reported, likely due to increased reactivity at extremely low pH levels. Although no significant formation of labelled NH₄⁺ or NO₃⁻ was observed, the low N recovery (74%) suggests gaseous losses, likely due to the potential occurrence of N₂O, potentially influenced by the parent material, though it was not detected in this study. These findings provide new insights into abiotic nitrogen retention, potentially explaining the low mineral N losses and high DON leaching observed in temperate rainforest ecosystems. They highlight the need to further explore the interplay between redox processes and organic matter chemistry. Field-scale studies are still needed to fully understand the reduction of NO₃⁻ to NO₂⁻ and its role in DON formation.